Methods and assays for treating subjects with shank3 deletion, mutation or reduced expression

ABSTRACT

Methods and assays are disclosed for treating subjects with 22q13 deletion syndrome or SHANK3 deletion or duplication, mutation or reduced expression, where the methods comprise administering to the subject insulin-like growth factor 1 (IGF-1), IGF-1-derived peptide or analog, growth hormone, an AMPAkine, a compound that directly or indirectly enhances glutamate neurotransmission, including by inhibiting inhibitory (most typically GABA) transmission, or an agent that activates the growth hormone receptor or the insulin-like growth factor 1 (IGF-1) receptor, or a downstream signaling pathway thereof.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of and claims priority of PCTInternational Patent Application No. PCT/US2011/000860, filed May 16,2011, which designates the United States of America, and claims thebenefit of U.S. Provisional Patent Application No. 61/395,775, filed May17, 2010, the contents of which are herein incorporated by reference intheir entirety.

STATEMENT OF GOVERNMENT SUPPORT

This invention was made with government support under grant numberMH093725 awarded by the National Institute of Mental Health. Thegovernment has certain rights in the invention.

FIELD OF THE INVENTION

The present invention relates generally to methods, and assays forcompounds, for treating subjects with 22q13 deletion syndrome or SHANK3deletion, duplication, mutation or reduced expression.

BACKGROUND OF THE INVENTION

Throughout this application various publications are referred to inshort form. Full citations for these references may be found at the endof the specification. The disclosures of these publications are herebyincorporated by reference in their entirety into the subject applicationto more fully describe the art to which the subject invention pertains.

22q13 Deletion Syndrome.

Chromosome 22q13 deletion syndrome, also known as Phelan-McDermidSyndrome, was first described in case reports in the early 90s,culminating in a review of the 24 published cases and 37 additionalcases by Phelan et al. (2001). The studies conclusively demonstratedthat individuals identified with 22q13 deletion syndrome had globaldevelopmental delay and absent or severely delayed expressive speech.Furthermore, the overwhelming majority of cases had hypotonia (97%) withnormal or accelerated growth (95%). The developmental delay isassociated with mental retardation typically in the mild-to-moderaterange. Other, less universal, features included large hands (>75%),dysplastic toenails (>75%), and decreased perspiration. Behaviorcharacteristics include mouthing or chewing non-food items (>75%),decreased perception of pain (>75%), and autism or autistic-like traits.Approximately 75% of individuals with a 22q13 deletion syndromediagnosis have either a 22 q terminal deletion (i.e., a chromosome breakin 22q with loss of the segment distal to the break), or an interstitialdeletion (i.e., two breaks within the same chromosome arm and loss ofthe intervening segment). The remaining 25% of individuals diagnosedwith 22q13.3 deletion syndrome had deletions resulting from anunbalanced translocation or other structural rearrangement, includingring 22.

Hypotonia, global developmental delay and speech deficits togetherrepresent some of the most consistent findings, each in >95% of allpatients. The hypotonia in newborns with the syndrome can be associatedwith weak cry, poor head control, and feeding difficulties leading tofailure to thrive. In terms of developmental delay, in addition to themental retardation noted above, there is also evidence for a delay tomajor milestones, such that, for example, the average age for rollingover is approximately eight months, for crawling approximately 16months, and for walking approximately three years. Poor muscle tone,lack of balance, and decreased upper body strength contribute to thedelay in walking and ultimately, gait is often broad-based and unsteady.Finally, while infants with the syndrome typically babble at theappropriate age and children may acquire a limited vocabulary, byapproximately age four years many children have significant deficits inthe ability to speak. With intensive therapy, the individuals with thesyndrome may have some speech and increase their vocabularies. It isinteresting to note that receptive communication skills are moreadvanced than expressive language skills as demonstrated by the abilityof affected children to follow simple commands, demonstrate humor, andexpress emotions.

Role of SHANK3 in 22q13 Deletion Syndrome.

Three lines of evidence implicated a single gene, SHANK3 (for SH3 andmultiple ankyrin repeat domains 3, also referred to as proline-richsynapse associated protein 2/PROSAP2), in 22q13 deletion syndrome.First, careful analysis of the extent of the deletion in independentcases indicated a small critical region encompassing SHANK3. Thus, ananalysis of 33 cases with various forms of monosomy of chromosome 22(include ring 22, which as noted above is phenotypical similarly to thedeletion syndrome) showed that the 12 with simple deletions haddeletions of variable in size (from 160 kb to 9 Mb), with a minimalcritical region responsible for the phenotype including SHANK3, ACR, andRABL2B (Luciani et al., 2003). Similarly, an analysis of 56 patientswith the syndrome again demonstrated a very variable size of thedeletion (130 kb to 9 Mb) with deletion of SHANK3 found in all casesexplicitly tested, including the smallest deletion, with the minimalregion encompassing the same three genes (Wilson et al., 2003).Remarkably, the severity of the behavioral phenotype was not correlatedwith the size of the deletion, indicating that haploinsufficiency ofjust one or more of these three genes was primarily responsible for thephenotype. Higher resolution studies have now identified patients witheven smaller deletions, which exclude ACR and RABL2B from the minimalregion, leaving only SHANK3 as the causal gene for the deletion syndrome(Bonaglia et al., 2011).

The second line of evidence was the demonstration of a recurrentbreakpoint in SHANK3 in some cases with 22q13 deletion syndrome. Thefirst report of a translocation with a breakpoint in SHANK3 associatedwith 22q13 deletion syndrome already made the point that disruption ofSHANK3 likely underlied the disorder (Bonaglia et al., 2001). This groupwent on the identify two additional cases (Bonaglia et al., 2006), bothwith a breakpoint within the same 15-bp repeat unit in the SHANK3 gene(which overlapped with another SHANK3 breakpoint described by Wong etal., 1997). The presence of recurrent disruptions in SHANK3 led to theconclusion that disruption of this one gene is sufficient for thegeneration of 22q13 deletion syndrome.

Role of SHANK3 in Autism Spectrum Disorders (ASD).

Mutations directly in SHANK3 also resulting in the main features of22q13 deletion syndrome represent the final line of evidence. Thus,while it has become increasingly recognized that 22q13 deletion syndromecan present with ASD and in fact 22q13 deletions are commonly associatedwith ASD in literature surveys (Vorstman et al., 2006), three recentstudies explored the separate question as to whether SHANK3 disruptionand mutations can be found in cohorts with apparently idiopathic ASD. Inthe first such study (Durand et al., 2007), SHANK3 was analyzed by bothFISH and by direct sequencing in as many as 227 individuals with ASD.Three variants were identified. First, an individual with a de novodeletion of SHANK3 was identified; this individual had autism (narrowlydefined), absent language, and moderate mental retardation. Second, apaternally inherited translocation was identified that resulted in adeletion of the 22q13 region (including SHANK3) in a girl with autismand severe language delay, and a duplication of the same region in herbrother with Asperger syndrome. Finally, Durand et al. (2007) identifiedtwo brothers with autism, severely impaired speech, and severe mentalretardation, which carried a single-base insertion in SHANK3. Theinsertion, which was maternal in origin (likely due to germlinemosaicism in the mother), resulted in a frameshift at the COOH-terminalof the protein that disrupts domains involved in Homer and cortactinbinding and the sterile alpha motif (SAM) domain involved in assembly ofthe SHANK3 platform. Overexpression of the mutant form in culturedhippocampal neurons did not lead to synaptic localization of theheterologous protein, in contrast to the wild-type SHANK3 protein.

In a follow up to Durand et al. (2007), Moessner et al. (2007), examinedboth sequence and SHANK3 gene dosage in 400 individuals with ASD. Twodeletions were identified, as well as 1 de novo mutation. Furthermore,an additional deletion was identified in two siblings from an additionalcollection. The mutation, found in a girl with autism, results in aQ321R change in the ankyrin repeat domain at the NH2 terminal of SHANK3.

In a third study, Gauthier et al. (2009) sequenced SHANK3 in 427 ASDsubjects and identified a de novo deletion at an intronic donor splicesite and a missense variant transmitted from an epileptic father.

A de novo splice site variant of the SHANK3 gene has also been reportedin a patient with mental retardation and severe language delay (Hamdanet al., 2011). In addition, Shank3 mutant mice display autistic-likebehaviours (Bozdagi et al. 2010; Bangash et al., 2011; Peca et al.,2011; Wang et al., 2011).

Remarkably, SHANK3 mutations can also result in schizophrenia, includingatypical schizophrenia associated with mental retardation and/or earlyonset as recently shown by Gauthier et al. (2010).

Altogether, these studies strongly support a role for disruptions ofSHANK3 in developmental delay and ASD. Clearly, haploinsufficiency ofSHANK3, caused either by a chromosomal abnormality or a mutation, canresult in a profound phenotype. Furthermore, even overexpression ofSHANK3 can result in developmental disorders (considering, for example,the case with Asperger syndrome and three copies of the SHANK3 locusreported in Durand et al., 2007 or the case with three copies and ADHDreported in Moessner et al., 2007). Recent, very large scale studies inclinical samples demonstrate that ca. 0.3% of patients with intellectualdisability referred to for chromosome microarray have a SHANK3 deletionor duplication (Cooper et al., 2011). With the advent of clinicalsequencing, point mutations in SHANK3 are also being identified in theclinical setting and evidence from research studies indicates a similarrate (ca. 0.3%) making SHANK3 deletions and mutations one of the morecommon monogenic causes of developmental delay syndromes, intellectualdisability and ASD.

Function of SHANK Proteins in the Structure of the Synapse.

The post-synaptic density (PSD) is an electron-dense structureunderlying the postsynaptic membrane in glutamatergic synapses in thecentral nervous system (Okabe, 2007). The PSD is most commonly found ondendritic spines of pyramidal neurons of the neocortex and hippocampusand Purkinje cells of the cerebellum, as well as on dendritic shafts atsites of contact with interneurons in the neocortex and hippocampus, aswell as motoneurons in the spinal cord. As such the PSD represents acritical organelle for glutamatergic transmission. It has been shownthat the SHANK proteins (including SHANK3) are a major part of the PSD.Multiple analytical approaches, including the characterization ofantibodies directed against PSD preparations, two-hybrid screens, gelelectrophoresis and mass spectrometry and other modern proteomicapproaches have placed the SHANK proteins in the PSD (reviewed inBoeckers, 2006 and Okabe, 2007). Moreover, recent quantitative methodshave estimated that there are about 300 individual SHANK molecules in asingle postsynaptic site, representing something in the order of 5% ofthe total protein molecules and total protein mass in the site (Sugiyamaet al., 2005). As it has been postulated that SHANK proteins maynucleate the protein framework for the PSD, a recent study examined theability of the sterile alpha motif (SAM) of SHANK3 to form polymers byself-association (Baron et al., 2006). As with other SAM domains (Qiaoand Bowie, 2005), the SAM domain of SHANK3 was able to self-associate,giving rise to large sheets of parallel fibers. These studies supportthe hypothesis that sheets of the SHANK proteins can form the scaffoldor platform onto which the PSD is constructed. Such a role for the SHANKproteins has led to them being called “master scaffolding proteins” ofthe PSD.

The SHANK Protein Interactome.

With the SHANK proteins (including SHANK3) forming a molecular platformonto which the PSD protein complex can be constructed, other proteinsand protein complexes of the PSD can associate with the SHANK platform.Of the various protein complexes associated with glutamatergic synapses,there is good evidence that the NMDA receptor complex (NRC), themetabotropic glutamate receptor complex (mGC), and the AMPA receptorcomplex (ARC) associate with the SHANK platform (see Boeckers, 2006).

The NRC (Husi et al., 2000), analyzed after isolation by affinitypurification, includes receptors, scaffolding proteins, signalingproteins, and cytoskeletal proteins. Amongst the scaffolding proteinsidentified in the NRC are the SHANK proteins, and it is thought thatNMDA receptors are anchored to the SHANK platform through the mediationof PSD-95 and SAPAP/GKAP (see Boeckers, 2006). Thus, NMDA receptors aretethered to the postsynaptic membrane by interaction with PDZ domains ofPSD-95, while the guanylate kinase domain of PSD-95 interacts with theSAPAP/GKAP proteins, which in turn bind to the SHANK proteins.

Similarly, mGC is linked to the SHANK platform, at least in part viaHomer. The mGC (Farr et al., 2004), analyzed after immunoisolation ofmGluR5 and associated molecules, includes SHANK and Homer proteins, bothof which have been previously associated with metabotropic glutamatereceptors using other methods. Homer proteins bind the cytoplasmicdomain of mGlu receptors (Brakeman et al., 1997) and couple mGlureceptors - - - and hence the mGC - - - to the SHANK platform (Tu etal., 1999). As SHANK proteins are able to bind to the IP3 receptor, thisinteraction also links mGlu receptors to the IP3 receptor (Sala et al.,2005).

Finally, the components of the ARC are bound to the SHANK platform.There is evidence for a direct interaction between the GluR1 AMPAreceptor and SHANK3 (Uchino et al., 2006). Moreover, there is evidencefor an indirect interaction in which transmembrane AMPA regulatoryprotein (TARP) subunits, including stargazin, bind both AMPA receptorsand PSD-95 (e.g., Bats et al., 2007). The interaction of AMPA receptorswith PSD-95 in turn allows for the linking of AMPA receptors with theSHANK platform via SAPAP/GKAP.

There are additional important interactions that involve the SHANKplatform, but even focusing on these three protein complexes, NRC, mGC,and ARC, it is clear that the SHANK proteins are critically involved inthe molecular architecture of glutamatergic synapses. Moreover, as SHANKproteins also interact with F-actin (the major cytoskeletal component ofspines) through cortactin (Naisbitt et al., 1999) and additionalmechanisms (see Boeckers, 2006), the SHANK platform is also likelyinvolved in the dynamic remodeling of glutamatergic synapses over shortand longer time frames (e.g., Hering and Sheng, 2003).

Modulation of SHANK3 Expression and Synapse Formation.

Overexpression of SHANK1 leads to increased spine size in neurons inculture (Sala et al., 2001). This effect, which could be furtherenhanced with the cotransfection of Homerl, also led to the recruitmentof Homer, PSD-95, and GKAP to the spines, along with glutamatereceptors, the IP3 receptor, and F-actin and bassoon, with enhancementof synaptic function, as measured electrophysiologically (Sala et al.,2001). More recent studies with SHANK3 support these conclusions(Roussignol et al., 2005). Thus, introduction of an siRNA constructinhibiting SHANK3 expression led to reduced number of spines inhippocampal neurons in culture. Furthermore, Roussignol et al. (2005)demonstrated that the introduction of SHANK3 into aspiny cerebellarneurons was sufficient to induce functional dendritic spines in thesecells, which then express functional NMDA and AMPA receptors.Altogether, these studies in cultured cells support a critical role forSHANK proteins in the development and function of the PSD and theglutamatergic synapse.

Recently, SHANK1 homozygous knockout mice were described which showedalterations in PSD thickness and PSD protein make-up, changes in spinemorphology, and decrease glutamatergic synaptic strength (but no changesin long term potentiation (LTP)) (Hung et al. 2008). These changes wereassociated with an increase in anxiety behavior, deficiencies onrotarod, impaired memory in a contextual fear task and in retention in aradial maze, but increased acquisition in the radial maze, confirming arole for SHANK proteins in glutamatergic transmission and behavior.

Regulation of SHANK3 Expression by Methylation.

Proper expression of SHANK3 is an important element of spine formationand brain development. Methylation of genes is one important means ofregulating expression. Interestingly, in a genome-wide analysis, SHANK3was identified as one of several genes where there was a clearrelationship between methylation status at CpG islands in the gene andexpression (Ching et al., 2005). The authors demonstrated that SHANK3 isexpressed in brain tissue, where the gene is predominantly unmethylated,and not expressed in lymphocytes, where the CpG island studied in theSHANK3 gene was nearly completely methylated.

The study of Ching et al. (2005) was followed by a more recent studythat looked in greater detail at SHANK3 as well as at the CpG islands inSHANK1 and SHANK2 (Beni et al., 2007). The authors identified 5 CpGislands in SHANK3 (one of which - - - identified by Beni et al. (2007)as CpG 4 - - - was the CpG island studied by Ching et al., 2005) and anequivalent number in SHANK1 and SHANK2. Only SHANK3 demonstratedtissue-specific methylation of CpG islands, with a relationship betweenmethylation and tissue-specific expression. These studies demonstratednot only that methylation at several of the CpG islands of SHANK3correlated with SHANK3 expression, but also that modulating themethylation of SHANK3 in cells in culture altered SHANK3 expression.Thus, treating primary neuronal cultures with methionine to increasemethylation resulted in decreased expression of SHANK3, while treatingHeLa cells with the demethylating agent 5-AdC resulted in decreasedmethylation of SHANK3 and increased expression of this gene in thesecells, which do not normally express SHANK3. Significantly, thedecreased expression of SHANK3 in primary neurons treated withmethionine was associated with decreased numbers of dendritic spines andwith decreased spine width, similar to what was observed by this samegroup with siRNA treatment of such cells (see above and Roussignol etal., 2005).

It has been shown that a proportion (0.5-1%) of children diagnosed withautism or autism spectrum disorders have deletions, duplications ormutations in SHANK3. While individuals with a diagnosis of 22q13deletion syndrome are relatively rare, autism and autism spectrumdisorders occur with a frequency of about 1 in 100 children. Consideringthis, as well as the rates of intellectual disability syndromes in thepopulation, it can be estimated that at least 1/6,000-1/16,000individuals will have deletions, duplications or mutations in SHANK3with associated phenotypes. This translates to ˜20-60,000 individuals inthe USA alone with life-long disability due to alterations in SHANK3expression. Thus, there is a compelling need for treatments for subjectswith 22q13 deletions or duplications or SHANK3 mutations. The presentinvention addresses this need.

SUMMARY OF THE INVENTION

The present invention provides methods for treating subjects with 22q13deletion syndrome or SHANK3 deletion or duplication, SHANK3 mutation orreduced expression of SHANK3, in need thereof, the methods comprisingadministering to the subject insulin-like growth factor 1 (IGF-1), anactive IGF-1 fragment including the tripeptide (1-3)IGF-1 or an analogthereof, growth hormone, or an AMPAkine, or another compound thatdirectly or indirectly enhances glutamate neurotransmission, includingby inhibiting inhibitory (most typically γ-aminobutyric acid (GABA))transmission, in an amount and manner effective to treat a subject with22q13 deletion syndrome or SHANK3 deletion or duplication, mutation orreduced expression.

The present invention further provides methods for treating subjectswith 22q13 deletion syndrome or SHANK3 deletion or duplication, mutationor reduced expression in need thereof, the methods comprisingadministering to the subject an agent that activates the growth hormonereceptor, or a downstream signaling pathway thereof, or the insulin-likegrowth factor 1 (IGF-1) receptor, or a downstream signaling pathwaythereof, or a downstream signaling pathway of (1-3)IGF-1, in an amountand manner effective to treat a subject with 22q13 deletion syndrome orSHANK3 deletion or duplication, mutation or reduced expression.

The present invention also provides methods for screening for agents fortreating subjects with 22q13 deletion syndrome or SHANK3 deletion orduplication, mutation or reduced expression, the methods comprisingdetermining whether or not the agent enhances long-term potentiation orincreases glutamate transmission, wherein an agent that enhanceslong-term potentiation or increases glutamate transmission is acandidate for treating a subject with 22q13 deletion syndrome or SHANK3deletion or duplication, mutation or reduced expression, whereas anagent that does not enhance long-term potentiation or increase glutamatetransmission is not a candidate for treating a subject with 22q13deletion syndrome or SHANK3 deletion or duplication, mutation or reducedexpression.

The present invention also provides methods for screening for agents fortreating subjects with 22q13 deletion syndrome or SHANK3 deletion orduplication, mutation or reduced expression, the methods comprisingdetermining whether or not the agent activates the growth hormonereceptor, or a downstream signaling pathway thereof, or the insulin-likegrowth factor 1 (IGF-1) receptor, or a downstream signaling pathwaythereof, or a downstream signaling pathway of (1-3)IGF-1, wherein anagent that activates the growth hormone receptor, or a downstreamsignaling pathway thereof, or the insulin-like growth factor 1 (IGF-1)receptor, or a downstream signaling pathway thereof, or a downstreamsignaling pathway of (1-3)IGF-1 is a candidate for treating a subjectwith 22q13 deletion syndrome or SHANK3 deletion or duplication, mutationor reduced expression, whereas an agent that does not activate thegrowth hormone receptor, or a downstream signaling pathway thereof, orthe insulin-like growth factor 1 (IGF-1) receptor, or a downstreamsignaling pathway thereof, or a downstream signaling pathway of(1-3)IGF-1 is not a candidate for treating a subject with 22q13 deletionsyndrome or SHANK3 deletion or duplication, mutation or reducedexpression.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A-1B. Long-term potentiation is impaired in Shank3 heterozygotes.Long-term potentiation (LTP) was induced either by (B) high frequencystimulus (HFS) (4×100 Hz, separated by 5 min) or (A) theta-burststimulus (TBS) (10 bursts of four pulses at 100 Hz separated by 200 ms)in hippocampal slices in mice. In both conditions, LTP as assessed byfield recordings of excitatory postsynaptic potential (EPSP) wasimpaired in the heterozygous animals (Het) compared to wildtype (WT).

FIG. 2A-2B. Basal synaptic transmission is reduced in Shank3-deficientmice. Mice with a targeted disruption of one copy of the Shank3 gene(“Shank3 heterozygotes”) and knockouts were compared to wild typelittermate controls. Both the input-output curve (figure) and theamplitude of miniature excitatory postsynaptic currents (EPSCs) (notshown) from hippocampal CA1 pyramidal neurons for Shank3 heterozygotesare significantly lower than those in control mice indicating areduction in basal transmission due to a postsynaptic effect. (A) Fieldexcitatory postsynaptic potential (fEPSP) slope versus stimulusintensity for wild type (WT), heterozygous (Het), and knockout (KO).Average slope of input-output function: WT (+/+), 1.38±0.3; Het (+/−),1.07±0.2; KO (−/−), 0.91±0.2, F2,21=7.30, p<0.01. (B) Both LTP inductionand maintenance is impaired in Shank3 knockouts, indicating a moresevere phenotype in the knockout mice. fEPSP slope versus time for WT,Het and KO. In the +/+control group, fEPSP slope recorded in area CA1significantly increased over baseline after TBS and was sustained for atleast 60 min (154.7±2.9% of baseline at 60 min, 159.3±2.6% at 40 minpost-TBS). In Shank3 −/− mice, the initial potentiation wassignificantly lower and decayed rapidly to baseline by 40 min(101.9±2.4% at 40 min post-TBS, N=4-7 mice per genotype, F(2,14)=85.2,p<0.001). Shank3+/−mice also showed reduced TBS-induced LTP but normalinitial potentiation.

FIG. 3A-3B. Both Shank3 heterozygous and homozygous mice do not showalteration in long-term depression. Long-term depression is inducedeither by (A) low frequency stimulus (LFS, 900 pulses at 1 Hz; 15 minduration) or (B) paired-pulse low frequency stimulus (PP-LFS, 1 Hz for20 min; 50 ms interstimulus interval), which is known to inducemGluR-dependent form of long-term depression. N=3 for each group. N=3for each group.

FIG. 4A-4B. Decrease in the AMPA component of fEPSP in SHANK3heterozygotes. (A) fEPSP slope versus fiber volley amplitude for theNMDA component of neurotransmission, carried out in the presence ofCNQX, a blocker of AMPA receptors. (B) fEPSP slope versus fiber volleyamplitude for the AMPA component of neurotransmission, carried out inthe presence of APV, a blocker of NMDA receptors (N=4 mice per genotype,two to three slices per mouse; P=0.001).

FIG. 5A-5C. Effects of (1-3)IGF-1 treatment on long-term potentiation atSchaffer collateral-CA1 synapses. (1-3)IGF-1 was administered daily viai.p. injections (0.01 mg/g body weight) starting at P13-15 andcontinuing for 2 weeks for electrophysiological recordings. (A)Hippocampal slices from wildtype (WT) and Shank3 heterozygous (Het) miceinjected with vehicle (saline, 0.01% bovine serum albumin (BSA)) or(1-3)IGF-1 were subjected to an LTP inducing stimulation, producinglong-lasting potentiation as shown by normalized field EPSP slope as afunction of time. Vehicle-treated heterozygotes showed reduced LTP,which was reversed by (1-3)IGF-1 (ANOVA, F(2,11)=8.98, p=0.007 at 90min. The inset shows representative EPSP traces at 90 min after LTPinduction from saline-injected (1) and (1-3)IGF-1-injected (2)heterozygous mice (scale bar: 0.5 mV, 10 ms). (B) Input-output curves,plotting field EPSP slopes (mV/ms) as a function of stimulation strength(mA) were significantly suppressed in slices from Shank3 heterozygousmice, but were not different from the wild type in heterozygous miceinjected with (1-3)IGF-1. (C) (1-3)IGF-1 peptide (same protocol used inthe treatment of heterozygote mice) treatment reversed the impairment inLTP in Shank3 KO mice. fEPSP versus time for KO, KO injected with IGF1peptide (N=2), and WT mice.

FIG. 6. IGF1 treatment activates PI3K-Akt pathway in the hippocampus.PI3K binds to AMPARs and is required to maintain AMPAR surfaceexpression during long-term potentiation. Figure shows the increase inthe phosphorylation of Akt (pAkt1) after IGF1 treatment in heterozygotemice, compared to the vehicle injected mice (n=3, P=0.0366, unpaired ttest). The data implicates the PI3K-Akt pathway in the beneficialeffects of (1-3)IGF-1.

FIG. 7. Effect of intranasal recombinant IGF-1 treatment on field EPSPin Shank3 heterozygotes (Het). Two-week old mice were anesthetized witha mixture of ketamine and xylazine. Recombinant human IGF-1 (rhIGF-1) orsaline was administered intranasally at 48 h intervals for a total of 10doses (15 μl solution containing 60 μg IGF-1 or vehicle per mouse wasgiven over 10-15 min period). N=2.

FIG. 8. Ampakine CX1837 restores long-term potentiation in Shank3heterozygous mice. Effects of CX1837 treatment on long-term potentiationat Schaffer collateral-CA1 synapses in Shank3 heterozygous mice.Ampakine CX1837 or HPCD vehicle is administered daily via i.p.injections (1.5 mg/kg body weight) starting at 2 weeks old and continuedfor 4 weeks for electrophysiological recordings.

FIG. 9. Effects of growth hormone treatment on long-term potentiation atSchaffer collateral-CA1 synapses in Shank3 heterozygous mice. Growthhormone is administered daily via i.p. injections (1 mg/kg body weight)starting at P13-15 and continued for 2 weeks for electrophysiologicalrecordings.

FIG. 10A-10B. Recombinant human IGF-1 (rhIGF-1) reverses deficits inlong-term potentiation and basal synaptic properties at Schaffercollateral-CA1 synapses in Shank3 heterozygous mice in a dose-dependentfashion. rhIGF-1 is administered daily via i.p. injections (120 or 240μg/kg body weight) starting at P13-15 and continued for 2 weeks forelectrophysiological recordings. A. LTP was induced with high-frequencystimulation and normalized field EPSP slope was plotted as a function oftime. Vehicle-treated heterozygotes showed reduced LTP, which wasreversed by the higher, but not the lower dose of rhIGF-1 (ANOVA,F(2,11)=14.39, p=0.002). The inset shows representative EPSP traces at90 min after LTP induction from saline-injected (1) and rhIGF-1-injected(2) heterozygous mice (scale bar: 0.5 mV, 10 ms). B. AMPA receptorresponses were assessed in the mice. Slices were incubated in thepresence of APV and mean field EPSP slope as a function of fiber volleyis shown for slices derived from wildtype (WT), Shank3 heterozygous(Het) mice, and Het injected with rhIGF-1. Deficits in AMPA receptorsignaling observed in Shank3 heterozygotes were reversed with 2-weekrhIGF-1 treatment.

FIG. 11. IGF-1 treatment reverses motor deficits in Shank3 heterozygousmice. Male wildtype (WT) and Shank3 heterozygous (Het) mice, treatedwith vehicle or recombinant human IGF-1 were tested for motorperformance and learning by measuring latencies to fall off a rotatingrod. Mice were challenged with three 2-minute trials (each separated by15 minutes) where the rotation was gradually increased from 0 to 45 rpm.Heterozygous mice injected with saline exhibit reduced latencies to fallcompared to wildtype mice. After IGF-1 treatment, heterozygous miceexhibit significantly longer latencies in comparison to vehicle-injectedmice of the same genotype.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a method for treating a subject with22q13 deletion syndrome or SHANK3 deletion or duplication, SHANK3mutation or reduced expression of SHANK3, in need thereof, the methodcomprising administering to the subject insulin-like growth factor 1(IGF-1), an active IGF-1 fragment comprising the tripeptide (1-3)IGF-1or an analog thereof, growth hormone, or an AMPAkine, or anothercompound that directly or indirectly enhances glutamateneurotransmission, including by inhibiting inhibitory (most typicallyGABA) transmission, in an amount and manner effective to treat a subjectwith 22q13 deletion syndrome or SHANK3 deletion or duplication, mutationor reduced expression. Reduced SHANK3 expression can be due, forexample, to abnormal methylation of the gene encoding SHANK3.

The invention thus provides a method for treating a subject with 22q13deletion syndrome or SHANK3 deletion or duplication, SHANK3 mutation orreduced expression of SHANK3, the method comprising administering to thesubject insulin-like growth factor 1 (IGF-1) or an active IGF-1 fragmentincluding the tripeptide (1-3)IGF-1 or an analog thereof, in an amountand manner effective to treat a subject with 22q13 deletion syndrome orSHANK3 deletion or duplication, mutation or reduced expression, whereinthe subject has autism spectrum disorder, autism, Asperger syndrome,pervasive developmental disorder, mental retardation, hypotonia, aspeech deficit, or developmental delay and/or defects.

The invention further provides a method for treating a subject withautism spectrum disorder, autism or Asperger syndrome comprisingadministering to the subject insulin-like growth factor 1 (IGF-1) or anactive IGF-1 fragment including the tripeptide (1-3)IGF-1 or an analogthereof, in an amount and manner effective to treat a subject withautism spectrum disorder, autism, or Asperger syndrome.

The present invention also provides a method for treating a subject with22q13 deletion syndrome or SHANK3 deletion or duplication, mutation orreduced expression in need thereof, the method comprising administeringto the subject an agent that activates the growth hormone receptor, or adownstream signaling pathway thereof, or the insulin-like growth factor1 (IGF-1) receptor, or a downstream signaling pathway thereof, or adownstream signaling pathway of (1-3)IGF-1, in an amount and mannereffective to treat a subject with 22q13 deletion syndrome or SHANK3deletion or duplication, mutation or reduced expression. As discussedherein, growth hormone stimulates production of IGF-1, and the maindownstream signaling pathways of the IGF-1 receptor are thephosphoinositide 3-kinase (PI3K), 3-phosphoinositide-dependent proteinkinase 1 (PDK), Akt, mammalian target of rapamycin (mTOR), andextracellular-signal-regulated kinase (ERK) pathways. Examples of suchagents include growth hormone, insulin-like growth factor 1 (IGF-1), thetripeptide (1-3)IGF-1 and analogs thereof.

As used herein, to treat a subject with 22q13 deletion syndrome orSHANK3 deletion or duplication, SHANK3 mutation or reduced expression ofSHANK3 means to alleviate a sign or symptom associated with 22q13deletion syndrome or SHANK3 deletion or duplication, mutation or reducedexpression. The syndrome is characterized by general hypotonia, motordeficits, absent to delayed speech, and global developmental delays.Individuals with a 22q13 deletion or SHANK3 mutation can suffer from arange of symptoms, with mild to very serious physical and behavioralcharacteristics. Possible symptoms include, but are not limited to,absent to severely delayed speech; hypotonia; increased tolerance topain; thin, flaky toenails; ptosis; poor thermoregulation; chewingnon-food items; teeth grinding; autistic behaviors; tongue thrusting;hair pulling; aversion to clothes; as well as other physical andbehavioral symptoms, including autism spectrum disorders and atypicalschizophrenia.

The present invention also provides a method for screening for agentsfor treating a subject with 22q13 deletion syndrome or SHANK3 deletionor duplication, mutation or reduced expression, the method comprisingdetermining whether or not the agent enhances long-term potentiation orincreases glutamate transmission, wherein an agent that enhanceslong-term potentiation or increases glutamate transmission is acandidate for treating a subject with 22q13 deletion syndrome or SHANK3deletion or duplication, mutation or reduced expression, whereas anagent that does not enhance long-term potentiation or increase glutamatetransmission is not a candidate for treating a subject with 22q13deletion syndrome or SHANK3 deletion or duplication, mutation or reducedexpression. The assay can be carried out, for example, using mice with adisruption of at least one copy of SHANK3 (e.g., Shank3 heterozygousmice).

The present invention also provides a method for screening for agentsfor treating a subject with 22q13 deletion syndrome or SHANK3 deletionor duplication, mutation or reduced expression, the method comprisingdetermining whether or not the agent activates the growth hormonereceptor, or a downstream signaling pathway thereof, or the insulin-likegrowth factor 1 (IGF-1) receptor, or a downstream signaling pathwaythereof, or a downstream signaling pathway of (1-3)IGF-1, wherein anagent that activates the growth hormone receptor, or a downstreamsignaling pathway thereof, or the insulin-like growth factor 1 (IGF-1)receptor, or a downstream signaling pathway thereof, or a downstreamsignaling pathway of (1-3)IGF-1 is a candidate for treating a subjectwith 22q13 deletion syndrome or SHANK3 deletion or duplication, mutationor reduced expression, whereas an agent that does not activate thegrowth hormone receptor, or a downstream signaling pathway thereof, orthe insulin-like growth factor 1 (IGF-1) receptor, or a downstreamsignaling pathway thereof, or a downstream signaling pathway of(1-3)IGF-1 is not a candidate for treating a subject with 22q13 deletionsyndrome or SHANK3 deletion or duplication, mutation or reducedexpression. Downstream signaling pathways of the IGF-1 receptor includethe PI3K, PDK, Akt, mTOR and ERK pathways.

The assays can be carried out, for example, using a brain slicepreparation, such as a hippocampal slice preparation, such as, forexample, described herein in Experimental Details. Preferably, the brainslice is from an animal, such as a mouse, with a disruption of at leastone copy of SHANK3 (e.g., a Shank3 heterozygous mouse).

Growth hormone (GH) is a protein based polypeptide hormone whichstimulates growth and cell reproduction and regeneration in humans andother animals. Growth hormone is synthesized, stored, and secreted bythe somatotroph cells in the anterior pituitary gland. Growth hormone isused clinically to treat children's growth disorders and adult growthhormone deficiency. Growth hormone stimulates production of IGF-1.Growth hormone can refer either to the natural hormone produced by thepituitary or biosynthetic growth hormone for therapy. Somatotropinrefers to the growth hormone produced naturally in animals, whereas theterm somatropin refers to growth hormone produced by recombinant DNAtechnology. In preferred embodiments, growth hormone is human growthhormone having the amino acid sequence (SEQ ID NO:1) (AccessionAAH90045)

1 matgsrtsll lafgllclpw lqegsafpti plsrlfdnam lrahrlhqla fdtyqefeea 61yipkeqkysf lqnpqtslcf sesiptpsnr eetqqksnle llrisllliq swlepvqflr 121svfanslvyg asdsnvydll kdleegiqtl mgrledgspr tgqifkqtys kfdtnshndd 181allknyglly cfrkdmdkve tflrivqcrs vegscgfor recombinant human growth hormone having the amino acid sequence (SEQID NO:2)

1 fptiplsrlf dnamlrahrl hqlafdtyqe feeayipkeq kysflqnpqt slcfsesipt 61psnreetqqk snlellrisl lliqswlepv qflrsvfans lvygasdsnv ydllkdleeg 121iqtlmgrled gsprtgqifk qtyskfdtns hnddallkny gllycfrkdm dkvetflriv 181qcrsvegscg f.Recombinant human growth hormone is available, e.g., from CellSciences®, Canton Mass. (Catalog No. CRH200A-C) (SEQ ID NO:2).

Insulin-like growth factor 1 (IGF-1), also known as somatomedin C ormechano growth factor, is a protein that is encoded by the IGF1 gene inhumans. IGF-1 is a hormone similar in molecular structure to insulin. Itplays an important role in childhood growth and continues to have ananabolic effect in adults. A synthetic analog of IGF-1, mecasermin, isused for the treatment of growth failure. IGF-1 is produced primarily bythe liver as an endocrine hormone as well as in target tissues in aparacrine/autocrine fashion. Production is stimulated by growth hormoneand can be retarded by undernutrition, growth hormone insensitivity,lack of growth hormone receptors, or failure of the downstream signalingpathway post growth hormone receptors including SHP2 and STAT5B. IGF-1has substantial human safety data and is approved for use in children.In preferred embodiment, IGF-1 is recombinant human IGF-1 (a 7.6 kDaprotein) having the amino acid sequence (SEQ ID NO:3)

1 gpetlcgael vdalqfvcgd rgfyfnkptg ygsssrrapq tgivdeccfr scdlrrlemy 61caplkpaksa.

The tripeptide (1-3)IGF-1 has the amino acid sequenceglycine-proline-glutamic acid (Gly-Pro-Glu or GPE). (1-3)IGF-1 can beobtained from Bachem (Torrance, Calif.) as H-Gly-Pro-Glu-OH (Catalog No.H-2468). The peptide has the advantage that it can penetrate theblood-brain barrier. Analogs of (1-3)IGF-1 that can be used include, butare not limited to, (1-3) IGF-1 amide, (1-3) IGF-1 stearate,Gly-Pro-Dglutamate, glycine-proline-threonine (Gly-Pro-Thr),glycine-glutamic acid-proline (Gly-Glu-Pro), glutamicacid-glycine-proline (Glu-Gly-Pro), and glutamic acid-proline-glycine(Glu-Pro-Gly).

AMPAkines are a class of compounds that strongly interact withglutamergic AMPA (α-amino-3-hydroxyl-5-methyl-4-isoxazolepropionic acid)receptors. Glutamergic AMPA receptors are non-NMDA-type ionotropictransmembrane receptors for glutamate that mediates fast synaptictransmission in the central nervous system (CNS). To date, fourstructural classes of AMPAkines have been developed: pyrrolidinederivatives of racetam drugs such as piracetam and aniracetam; CX-seriesof drugs encompassing a range of benzoylpiperidine andbenzoylpyrrilidine structures; benothiazide derivatives such ascyclothiazide and IDRA-21; and biarylpropylsulfonamides such asLY-392,098, LY-404,187, LY-451,646, and LY-503,430. AMPAkines bind toglutamergic AMPA receptors, boosting the activity of glutamate, aneurotransmitter, and making it easier to encode memory and learning.Some AMPAkines may increase levels of trophic factors such asbrain-derived neurotrophic factor. Preferred AMPAkines include CXAMPAkines (Cortex Pharmaceuticals, Inc., Irvine, Calif.), such as forexample CX-516 (Ampalex), CX-546, CX-614, CX-691 (Farampator), CX-717,CX-701, CX-1739, CX-1763 and CX-1837 (see, e.g., U.S. Pat. Nos.5,650,409, 5,736,543, 5,985,871, 6,166,008, 6,313,115, and 7,799,913,and U.S. Patent Application Publications No. 2002/0055508, 2010/0041647,2010/0173903, 2010/0267728, 2010/02866177, and 2011/0003835, thecontents of which are herein incorporated by reference).

Compounds that directly or indirectly enhance glutamateneurotransmission including, for example, by inhibiting inhibitory (mosttypically GABA) transmission, include, for example, glycine transporter1 (GLYT1) inhibitors, brain-derived neurotrophic factor (BDNF), andcyclothiazide. Cyclothiazide acts both on AMPA receptors and GABA(A)receptors. GLYT1 inhibitors are a functional class of compounds andinclude compounds that act as GABA(A) receptor negative allostericmodulators and inhibitors. Specific GLYT1 inhibitors include, forexample, NFPS, Org 24461, and sarcosine.

IGF-1 interacts with its receptor (IGF-1R) to initiate downstreamresponses such as proliferation and differentiation. The IGF-1R is atransmembrane receptor that is activated by IGF-1 and by the relatedgrowth factor IGF-2. It belongs to the large class of tyrosine kinasereceptors and mediates the effects of IGF-1. Tyrosine kinase receptors,including the IGF-1R, mediate their activity by causing the addition ofa phosphate group to particular tyrosines on certain proteins within acell. This addition of phosphate induces what are called “cellsignaling” cascades - and the usual result of activation of the IGF-1Ris survival and proliferation in mitosis-competent cells, and growth(hypertrophy) in tissues such as skeletal muscle and cardiac muscle.IGF-1R activates several downstream signaling pathways. The maindownstream signaling pathways of IGF-1R are the PI3K, PDK, Akt, mTOR andERK pathways.

Phosphoinositide 3-kinases (PI 3-kinases or PI3Ks) are a family ofenzymes involved in cellular functions such as cell growth,proliferation, differentiation, motility, survival and intracellulartrafficking. PI3Ks are downstream of IGF-1R and interact with the IRS(Insulin receptor substrate) in order to regulate cell function uptakethrough a series of phosphorylation events. The phosphoinositol-3-kinasefamily is divided into three different classes: Class I, Class II, andClass III. The classifications are based on primary structure,regulation, and in vitro lipid substrate specificity. PI3K has also beenimplicated in Long-term potentiation (LTP). PI3Ks are necessary for thesurvival of progenitors and mature oligodendrocytes and for theIGF-1-mediated cell survival, proliferation, and protein synthesis.

AKT protein family, which members are also called protein kinases B(PKB) plays an important role in mammalian cellular signaling. Inhumans, there are three genes in the “Akt family”: Akt1, Akt2, and Akt3.These genes code for enzymes that are members of theserine/threonine-specific protein kinase family. Akt1 is involved incellular survival pathways, by inhibiting apoptotic processes. Akt1 isalso able to induce protein synthesis pathways, and is therefore a keysignaling protein in the cellular pathways that lead to skeletal musclehypertrophy, and general tissue growth. Akt2 is an important signalingmolecule in the insulin signaling pathway. It is required to induceglucose transport. Akt can be phosphorylated by PDK1 and mTORC2. Besidedownstream effectors of PI3K, Akt can be activated in a PI3K-independentmanner. Akt2 is required for the insulin-induced translocation ofglucose transporter 4 (GLUT4) to the plasma membrane. Glycogen synthasekinase 3 (GSK-3) could be inhibited upon phosphorylation by Akt, whichresults in promotion of glycogen synthesis. Akt inhibitors anddominant-negative Akt expression can block IGF-1 stimulated proteinsynthesis in oligodendrocyte progenitors.

3-phosphoinositide dependent protein kinase-1 (PDK1) is a protein whichin humans is encoded by the PDPK1 gene and is a master kinase crucialfor the activation of AKT/PKB and many other AGC kinases including PKC,S6K, and SGK. An important role for PDK1 is in the signalling pathwaysactivated by several growth factors and hormones including insulinsignalling. PDK1 functions downstream of PI3K through PDK1's interactionwith membrane phospholipids including phosphatidylinositols,phosphatidylinositol (3,4)-bisphosphate and phosphatidylinositol(3,4,5)-trisphosphate. PI3K indirectly regulates PDPK1 byphosphorylating phosphatidylinositols which in turn generatesphosphatidylinositol (3,4)-bisphosphate and phosphatidylinositol(3,4,5)-trisphosphate.

The mammalian target of rapamycin (mTOR), also known as mechanistictarget of rapamycin or FK506 binding protein 12-rapamycin associatedprotein 1 (FRAP1), is a protein which in humans is encoded by the FRAP1gene. mTOR is a serine/threonine protein kinase that regulates cellgrowth, cell proliferation, cell motility, cell survival, proteinsynthesis, and transcription. mTOR integrates the input from upstreampathways, including insulin, growth factors (such as IGF-1 and IGF-2),and mitogens. mTOR also senses cellular nutrient and energy levels andredox status.

Extracellular-signal-regulated kinases (ERKs), or classical MAP kinases,are widely expressed protein kinase intracellular signalling moleculesthat are involved in functions including the regulation of meiosis,mitosis, and postmitotic functions in differentiated cells. Manydifferent stimuli, including growth factors (such as IGF-1 and IGF-2),cytokines, virus infection, ligands for heterotrimeric G protein-coupledreceptors, transforming agents, and carcinogens, activate the ERKpathway.

The 22q13 deletion syndrome or SHANK3 deletion or duplication, mutationor reduced expression can be treated by local or systemic administrationof the IGF-1, IGF-1-derived peptide or analog, growth hormone, AMPAkine,or other compound that directly or indirectly enhances glutamateneurotransmission, including by inhibiting inhibitory (most typicallyGABA) transmission, or other therapeutic agent. Local treatment maycomprise intramuscular or intratissue injection. Systemic treatment maycomprise enteral or intravenous methods. The IGF-1, IGF-1-derivedpeptide or analog, growth hormone, AMPAkine, or other compound thatdirectly or indirectly enhances glutamate neurotransmission, includingby inhibiting inhibitory (most typically GABA) transmission, or agentmay be administered in a pharmaceutical composition comprising theIGF-1, IGF-1-derived peptide or analog, growth hormone, AMPAkine, orother compound that directly or indirectly enhances glutamateneurotransmission, including by inhibiting inhibitory (most typicallyGABA) transmission, or agent in a pharmaceutically acceptable carrier.

The pharmaceutically acceptable carrier must be compatible with theIGF-1, IGF-1-derived peptide or analog, growth hormone, AMPAkine, orother compound that directly or indirectly enhances glutamateneurotransmission, including by inhibiting inhibitory (most typicallyGABA) transmission, or agent, and not deleterious to the subject.Examples of acceptable pharmaceutical carriers includecarboxymethylcellulose, crystalline cellulose, glycerin, gum arabic,lactose, magnesium stearate, methylcellulose, powders, saline, sodiumalginate, sucrose, starch, talc, and water, among others. Formulationsof the pharmaceutical composition may conveniently be presented in unitdosage and may be prepared by any method known in the pharmaceuticalart. For example, the IGF-1, IGF-1-derived peptide or analog, growthhormone, AMPAkine, or other compound that directly or indirectlyenhances glutamate neurotransmission, including by inhibiting inhibitory(most typically GABA) transmission, or agent may be brought intoassociation with a carrier or diluent, as a suspension or solution.Optionally, one or more accessory ingredients, such as buffers,flavoring agents, surface-active ingredients, and the like, may also beadded. The choice of carriers will depend on the method ofadministration. The pharmaceutical composition can be formulated foradministration by any method known in the art, including but not limitedto, intravenously and intracranially.

The amount of IGF-1, IGF-1-derived peptide or analog, growth hormone,AMPAkine, or other compound that directly or indirectly enhancesglutamate neurotransmission, including by inhibiting inhibitory (mosttypically GABA) transmission, or agent therapeutically necessary willdepend on the severity of the 22q13 deletion syndrome or SHANK3 mutationas well as the manner of administration of the IGF-1, IGF-1-derivedpeptide or analog, growth hormone, AMPAkine, or other compound thatdirectly or indirectly enhances glutamate neurotransmission, includingby inhibiting inhibitory (most typically GABA) transmission, or agent.One skilled in the art can easily determine the amount and manner ofadministration of IGF-1, IGF-1-derived peptide or analog, growthhormone, AMPAkine, or other compound that directly or indirectlyenhances glutamate neurotransmission, including by inhibiting inhibitory(most typically GABA) transmission, or agent necessary.

According to the method of the present invention, the IGF-1,IGF-1-derived peptide or analog, growth hormone, AMPAkine, or othercompound that directly or indirectly enhances glutamateneurotransmission, including by inhibiting inhibitory (most typicallyGABA) transmission, or other therapeutic agent may be administered to asubject by any known procedure including, but not limited to, oraladministration, parenteral administration, transdermal administration,intranasal administration, and administration through an osmoticmini-pump.

For oral administration, the formulation of the IGF-1, IGF-1-derivedpeptide or analog, growth hormone, AMPAkine, or other compound thatdirectly or indirectly enhances glutamate neurotransmission, includingby inhibiting inhibitory (most typically GABA) transmission, or othertherapeutic agent may be presented as capsules, tablets, powder,granules, or as a suspension. The formulation may have conventionaladditives, such as lactose, mannitol, corn starch, or potato starch. Theformulation may also be presented with binders, such as crystallinecellulose, cellulose derivatives, acacia, corn starch, or gelatins.Additionally, the formulation may be presented with disintegrators, suchas corn starch, potato starch, or sodium carboxymethylcellulose. Theformulation also may be presented with dibasic calcium phosphateanhydrous or sodium starch glycolate. Finally, the formulation may bepresented with lubricants, such as talc or magnesium stearate.

For a parenteral administration, the IGF-1, IGF-1-derived peptide oranalog, growth hormone, AMPAkine, or other compound that directly orindirectly enhances glutamate neurotransmission, including by inhibitinginhibitory (most typically GABA) transmission, or other therapeuticagent may be combined with a sterile aqueous solution which ispreferably isotonic with the blood of the subject. Such a formulationmay be prepared by dissolving a solid active ingredient in watercontaining physiologically-compatible substances, such as sodiumchloride, glycine, and the like, and having a buffered pH compatiblewith physiological conditions, so as to produce an aqueous solution,then rendering said solution sterile. The formulations may be present inunit or multi-dose containers, such as sealed ampoules or vials. Theformulation may be delivered by any mode of injection, including,without limitation, epifascial, intrasternal, intravascular,intravenous, parenchymatous, or subcutaneous.

For transdermal administration, the IGF-1, IGF-1-derived peptide oranalog, growth hormone, AMPAkine, or other compound that directly orindirectly enhances glutamate neurotransmission, including by inhibitinginhibitory (most typically GABA) transmission, or other therapeuticagent may be combined with skin penetration enhancers, such as propyleneglycol, polyethylene glycol, isopropanol, ethanol, oleic acid,N-methylpyrrolidone, and the like, which increase the permeability ofthe skin to the IGF-1, IGF-1-derived peptide or analog, growth hormone,AMPAkine, or other compound that directly or indirectly enhancesglutamate neurotransmission, including by inhibiting inhibitory (mosttypically GABA) transmission, or other therapeutic agent. The IGF-1,IGF-1-derived peptide or analog, growth hormone, AMPAkine, or othercompound that directly or indirectly enhances glutamateneurotransmission, including by inhibiting inhibitory (most typicallyGABA) transmission, or other therapeutic agent compositions also may befurther combined with a polymeric substance, such as ethylcellulose,hydroxypropyl cellulose, ethylene/vinylacetate, polyvinyl pyrrolidone,and the like, to provide the composition in gel form, which may bedissolved in solvent such as methylene chloride, evaporated to thedesired viscosity, and then applied to backing material to provide apatch.

The IGF-1, IGF-1-derived peptide or analog, growth hormone, AMPAkine, orother compound that directly or indirectly enhances glutamateneurotransmission, including by inhibiting inhibitory (most typicallyGABA) transmission, or other therapeutic agent may also be released ordelivered from an osmotic mini-pump. The release rate from an elementaryosmotic mini-pump may be modulated with a microporous, fast-response geldisposed in the release orifice. An osmotic mini-pump would be usefulfor controlling the release of, or targeting delivery of, the IGF-1,IGF-1-derived peptide or analog, growth hormone, AMPAkine, or othercompound that directly or indirectly enhances glutamateneurotransmission, including by inhibiting inhibitory (most typicallyGABA) transmission, or other therapeutic agent.

Long-term potentiation (LTP) is a long-lasting enhancement in signaltransmission between two neurons that results from stimulating themsynchronously. It is one of several phenomena underlying synapticplasticity, the ability of chemical synapses to change their strength.LTP shares many features with long-term memory, making it an attractivecandidate for a cellular mechanism of learning. For example, LTP andlong-term memory are triggered rapidly, each depends upon the synthesisof new proteins, each has properties of associativity, and each can lastfor many months. LTP may account for many types of learning, from therelatively simple classical conditioning present in all animals, to themore complex, higher-level cognition observed in humans. At a cellularlevel, LTP enhances synaptic transmission. It improves the ability oftwo neurons, one presynaptic and the other postsynaptic, to communicatewith one another across a synapse.

Chemical synapses are functional connections between neurons throughoutthe nervous system. In a typical synapse, information is largely passedfrom the first (presynaptic) neuron to the second (postsynaptic) neuronvia a process of synaptic transmission. Through experimentalmanipulation, a non-tetanic stimulus can be applied to the presynapticcell, causing it to release a neurotransmitter such as glutamate ontothe postsynaptic cell membrane. There, glutamate binds to receptors suchas AMPA receptors (AMPARs) embedded in the postsynaptic membrane. TheAMPA receptor is one of the main excitatory receptors in the brain, andis responsible for most of its rapid, moment-to-moment excitatoryactivity. Glutamate binding to the AMPA receptor triggers the influx ofpositively charged sodium ions into the postsynaptic cell, causing ashort-lived depolarization called the excitatory postsynaptic potential(EPSP). Extracellular-signal-regulated kinases (ERKs) play a role inlate LTP, where gene expression and protein synthesis is brought aboutby the persistent activation of protein kinases activated during earlyLTP. ERK phosphorylates a number of cytoplasmic and nuclear moleculesthat ultimately result in the protein synthesis and morphologicalchanges observed in late LTP. ERK-mediated changes in transcriptionfactor activity may trigger the synthesis of proteins that underlie themaintenance of L-LTP.

An excitatory postsynaptic potential (EPSP) is a temporarydepolarization of postsynaptic membrane potential caused by the flow ofpositively charged ions into the postsynaptic cell as a result ofopening of ligand-sensitive channels. ESPSs in living cells are causedchemically. When an active presynaptic cell releases neurotransmittersinto the synapse, some bind to receptors on the postsynaptic cell. Manyof these receptors contain an ion channel capable of passing positivelycharged ions either into or out of the cell. The depolarizing currentcauses an increase in membrane potential, the ESPS. The amino acidglutamate is the neurotransmitter most often associated with EPSPs.

Glutamate is the most abundant excitatory neurotransmitter in thevertebrate nervous system. At chemical synapses, glutamate is stored invesicles. Nerve impulses trigger release of glutamate from thepre-synaptic cell. In the opposing post-synaptic cell, glutamatereceptors, such as the NMDA receptor, bind glutamate and are activated.Because of its role in synaptic plasticity, glutamate is involved incognitive functions like learning and memory in the brain. The form ofplasticity known as long-term potentiation takes place at glutamatergicsynapses in the hippocampus, neocortex, and other parts of the brain.Glutamate does not work only as a point to point transmitter but alsothrough spill-over synaptic crosstalk between synapses in whichsummation of glutamate released from neighboring synapse createsextrasynaptic signaling/volume transmission.

Glutamate transporters are found in neuronal and glial membranes. Theyrapidly remove glutamate from the extracellular space. In brain injuryor disease, they can work in reverse and excess glutamate can accumulateoutside cells. This process causes calcium ions to enter cells via NMDAreceptor channels, leading to neuronal damage and eventual cell death,and is called excitotoxicity.

The subject can be any mammal, in particular a human. The subject canhave, for example, one or more of autism, Asperger syndrome, autismspectrum disorder, pervasive developmental disorder, mental retardation,hypotonia, speech deficits and developmental delay and/or defects.

EXPERIMENTAL DETAILS

Brief Experimental Procedures:

Hippocampal slices (350 μm) are prepared from 1-3 month old Shank3heterozygous, Shank3 knockout, and wildtype littermates, treated with(1-3) IGF-1 peptide, full-length IGF1, growth hormone, AMPAkine orsaline or other appropriate control. Slices are perfused with Ringer'ssolution, bubbled with 95% O₂/5% CO₂, at 32° C., during extracellularrecordings. Baseline of field excitatory postsynaptic potentials(fEPSPs) recorded from stratum radiatum in area CA1, evoked bystimulation of the Schaffer collateral-commissural afferents withbipolar tungsten electrodes placed into area CA3. Long-term potentiation(LTP) is induced either by a high-frequency stimulus (four trains of 100Hz, 1 s stimulation separated by 5 min), or by theta-burst stimulation(TBS) (10 bursts of four pulses at 100 Hz separated by 200 ms), with asuccess rate >90% for control and genetically-modified animals with allstimulation protocols. To induce long-term depression (LTD), Schaffercollaterals were stimulated by a low frequency stimulus (900 pulses at 1Hz for 15 min) or paired-pulse low frequency stimulus. LTD was inducedwith a success rate >90% for control animals. In order to examine Aktphosphorylation hippocampus and cortex are dissected from the otherhemisphere from Shank3 heterozygous and wildtype littermates, used tomake slices and are immediately snap-frozen on dry ice. Western blotanalysis is performed for total- and phospho-Akt levels. Results arepresented in FIGS. 1-10. Mice were also tested for motor performance andlearning by measuring latencies to fall off a rotating rod. Mice werechallenged with three 2-minute trials (each separated by 15 minutes)where the rotation was gradually increased from 0 to 45 rpm, and resultsare presented in FIG. 11.

A mouse with hemizygous loss of full-length Shank3 (Bozdagi et al.,2010) was used to investigate whether IGF-1 could reverse synapticdeficits in a preclinical model. Tests were first made with an activepeptide of IGF-1 ((1-3)IGF-1), which has been shown to cross theblood-brain barrier (O'Kusky et al., 2000) and rescue Rett syndromesymptoms in Mecp2-deficient mice. Intraperitoneal injection at 10μg/g/day for 2 weeks restored normal hippocampal LTP in Shank3heterozygous mice (FIG. 5a ). While LTP was significantly reduced invehicle treated heterozygotes, heterozygous mice treated with IGF-1showed a complete rescue. Similarly, LTP at 90 minutes after inductionwas significantly (P=0.007) reduced in vehicle-treated heterozygotes,compared to wildtype littermates, but not when comparing peptide treatedheterozygotes to wildtype animals. In addition, the significantly(P=0.029) reduced input-output (I/O) function observed in heterozygotes[obtained by plotting field excitatory postsynaptic potential (fEPSP)slope versus stimulus intensity], was reversed after 2 weeksadministration of active peptide of IGF-1 (FIG. 5b ), indicating thatdeficits in synaptic transmission are rescued by (1-3)IGF-1.

IGF-1 has been approved for clinical use as a recombinant full-lengthprotein. Full-length IGF-1 enters the CNS through an interaction withlipoprotein-related receptor 1 (LRP1), after activity dependent cleavageof IGF binding protein-3 (IGFBP-3) by matrix metalloproteinase-9 (MMP9)(Nishijima et al., 2010). To investigate whether peripherallyadministered full-length IGF-1 could also reverse synaptic deficits,IGF-1 was administered by intraperitoneal injection at 240 μg/kg/day,starting at P13-15 and continuing for 2 weeks. This dose was chosenbecause it represents the maximum dose according to the current FDAlabel for IGF-1. This treatment was also effective in rescuing defectiveLTP in heterozygous mice (FIG. 10a ). Furthermore, specific deficits inthe AMPA receptor component of I/O function (Bozdagi et al., 2010) werereversed by this treatment (FIG. 10b ). Mean slope of the I/O functionwas 0.625±0.08 for wildtype; 0.31±0.045 for Shank3 heterozygous and0.618±0.075 for IGF-1 injected heterozygous mice (comparison betweenheterozygotes and IGF-1-injected heterozygotes, p=0.004). Importantly,lower dose IGF-1 (120 μg/kg/day for 2 weeks) was not associated withsignificant reversal of deficits in LTP (FIG. 10a ), showing adose-response effect and providing preclinical dosing information.

Phelan-McDermid syndrome frequently presents with hypotonia and at leasttransient motor deficits, and subtle motor deficits have been observedin Shank3-heterozygous mice (Bozdagi et al., 2010). To determine whethertreatment with full-length IGF-1 may improve motor performance inShank3-deficient mice, male heterozygous mice were treated with eithervehicle or IGF-1 (240 μg/kg/day for 2 weeks). Significant enhancement ofmotor performance was observed following treatment (FIG. 11).

To date, pharmacological treatments for ASD and other neurodevelopmentaldisorders are primarily ameliorative, focusing on managing associatedsymptoms such as anxiety, aggression, repetitive behaviors, or epilepsy(King et al., 2006). Pharmacological treatments addressing “core”deficits, such as cognitive impairments, social deficits, and absent ordelayed speech, do not yet exist. Recently, however, the field has begunto see the evaluation of therapies targeted to etiology (“personalizedmedicine”) - - - each arising from the basic analysis of modelsystems - - - in neurodevelopmental disorders including Fragile Xsyndrome, tuberous sclerosis, and Rett syndrome (Bear et al., 2004;Ehninger et al., 2009; Tropea et al., 2009).

It is interesting to note that IGF-1 activates the mTOR/Akt pathway asthis has been implicated in other neurodevelopmental disorders(Veenstra-Vanderweele et al., 2012). It was therefore predicted thatphospho-Akt/Akt ratios would be increased after IGF-1 treatment, andthis is what was observed in studies in hippocampal lysates (0.36±0.03and 0.55±0.04 in Shank3 heterozygous mice injected for 2 weeks withvehicle [n=8] or full-length IGF-1 protein [n=10], respectively;1-tailed p=0.040).

Loss of one functional copy of the SHANK3 gene, through either mutationor deletion, is found in about 0.5% of ASD (Abrahams and Geschwind,2008) and in about 0.3% of the developmentally delayed population(Cooper et al., 2011). As such it represents one of the more frequentcauses of these disorders and a significant health burden. In addition,there is emerging evidence that the SHANK3 pathway may play a role inother neurodevelopmental disorders, as evidenced by large-scaleproteomic and gene expression studies (Darnell et al., 2011; Sakai etal., 2011). Even more broadly, deficits in proteins associated with thepostsynaptic density, which is in no small degree sculpted by SHANK3(Roussignol et al., 2005), are associated with neurodevelopmentaldisorders (Laumonnier et al., 2007). Mutations in SHANK3 are alsoassociated with schizophrenia (Gauthier et al., 2010). This indicatesthat therapies for SHANK3 deficiency and synaptic development representimportant targets that can have broad positive impact inneurodevelopmental disorders (Melom and Littleton, 2011). These resultsshow that IGF-1, approved for use in children, can lead to functionalimprovements in a mouse model of ASD and developmental delay,representing an important preclinical step.

REFERENCES

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1. A method for treating a subject with 22q13 deletion syndrome orSHANK3 deletion or duplication, SHANK3 mutation or reduced expression ofSHANK3, the method comprising administering to the subject insulin-likegrowth factor 1 (IGF-1), an active IGF-1 fragment including thetripeptide (1-3)IGF-1 or an analog thereof, growth hormone, an AMPAkine,or a compound that enhances glutamate neurotransmission, in an amountand manner effective to treat a subject with 22q13 deletion syndrome orSHANK3 deletion or duplication, mutation or reduced expression. 2.-4.(canceled)
 5. The method of claim 1, wherein the analog of (1-3)IGF-1 isselected from the group consisting of (1-3)IGF-1 amide, (1-3)IGF-1stearate, Gly-Pro-D-glutamate, glycine-proline-threonine (Gly-Pro-Thr),glycine-glutamic acid-proline (Gly-Glu-Pro), glutamicacid-glycine-proline (Glu-Gly-Pro), and glutamic acid-proline-glycine(Glu-Pro-Gly).
 6. The method of claim 1, wherein IGF-1, IGF-1-derivedpeptide or analog, growth hormone, AMPAkine, compound that enhancesglutamate neurotransmission, or agent is administered locally.
 7. Themethod of claim 1, wherein IGF-1, IGF-1-derived peptide or analog,growth hormone, AMPAkine, compound that enhances glutamateneurotransmission, or agent is administered systemically. 8-13.(canceled)
 14. The method of claim 1, wherein the subject is human. 15.The method of claim 14, wherein the subject has autism, Aspergersyndrome, autism spectrum disorder, pervasive developmental disorder,mental retardation, hypotonia, speech deficits, or a developmental delayand/or defect.
 16. The method of claim 1, wherein IGF-1, IGF-1-derivedpeptide or analog, growth hormone, AMPAkine, compound that enhancesglutamate neurotransmission, or agent alleviates one or more ofhypotonia; a motor deficit; absent speech; increased tolerance to pain;thin, flaky toenails; poor thermoregulation; chewing non-food items;teeth grinding; autistic behaviors; tongue thrusting; hair pulling; andaversion to clothes.
 17. The method of claim 1, wherein the compoundthat enhances glutamate neurotransmission inhibits an inhibitoryneurotransmitter.
 18. The method of claim 17, wherein the inhibitoryneurotransmitter is GABA.
 19. A method for treating a subject with 22q13deletion syndrome or SHANK3 deletion or duplication, SHANK3 mutation orreduced expression of SHANK3, the method comprising administering to thesubject insulin-like growth factor 1 (IGF-1) or an active IGF-1 fragmentincluding the tripeptide (1-3)IGF-1 or an analog thereof, in an amountand manner effective to treat a subject with 22q13 deletion syndrome orSHANK3 deletion or duplication, mutation or reduced expression, whereinthe subject has autism spectrum disorder, autism, Asperger syndrome,pervasive developmental disorder, mental retardation, hypotonia, aspeech deficit, or a developmental delay and/or defect.
 20. (canceled)21. A method for treating a human subject with Phelan-McDermid Syndromecomprising administering insulin-like growth factor 1 (IGF-1) to thehuman subject in an amount and manner effective to ameliorate delayedspeech in a human subject with Phelan-McDermid Syndrome.